Hepatic transglutaminase has been previously shown to catalyze the coupling of many primary amines to proteins. This enzyme is also able to catalyze the cross-linking of certain peptide chains. In the present study, this enzyme in the cytosol fraction of rabbit liver in the presence of calcium couples amines, such as putrescine, to endogenous proteins. The enzyme also cross-links endogenous proteins in the cytosol fraction to yield proteins with a very high molecular weight. The enzyme was isolated and purified from the cytosol fraction. The purified enzyme was able to cross-link proteins of purified rabbit liver plasma membranes. Very high concentrations of amines were required to inhibit this reaction. It is suggested that a physiological role of this enzyme might be the cross-linking of intracellular proteins and that this reaction might be regulated by calcium.